Conventional chemotherapy in malignant pleural mesothelioma (MPM) has minimal impact on patient survival due to the supposed chemoresistance of cancer stem cells (CSCs). We sought to identify a sub-population of chemoresistant cells by using putative CSC markers, aldehyde dehydrogenase (ALDH) and CD44 in three MPM cell lines; H28, H2052 and Meso4.
Cortes-Dericks et al BMC Cancer 2014, 14:304 http://www.biomedcentral.com/1471-2407/14/304 RESEARCH ARTICLE Open Access Cisplatin-resistant cells in malignant pleural mesothelioma cell lines show ALDHhighCD44+ phenotype and sphere-forming capacity Lourdes Cortes-Dericks, Laurene Froment, Ruben Boesch, Ralph Alexander Schmid* and Golnaz Karoubi Abstract Background: Conventional chemotherapy in malignant pleural mesothelioma (MPM) has minimal impact on patient survival due to the supposed chemoresistance of cancer stem cells (CSCs) We sought to identify a sub-population of chemoresistant cells by using putative CSC markers, aldehyde dehydrogenase (ALDH) and CD44 in three MPM cell lines; H28, H2052 and Meso4 Methods: The Aldefluor assay was used to measure ALDH activity and sort ALDHhigh and ALDHlow cells Drug-resistance was evaluated by cell viability, anchorage-independent sphere formation, flow-cytometry and qRT-PCR analyses Results: The ALDHhigh - and ALDHlow -sorted fractions were able to demonstrate phenotypic heterogeneity and generate spheres, the latter being less efficient, and both showed an association with CD44 Cis- diamminedichloroplatinum (II) (cisplatin) treatment failed to reduce ALDH activity and conferred only a short-term inhibition of sphere generation in both ALDHhigh and ALDHlow fractions of the three MPM cell lines Induction of drug sensitivity by an ALDH inhibitor, diethylaminobenzaldehyde (DEAB) resulted in significant reductions in cell viability but not a complete elimination of the sphere-forming cells, suggestive of the presence of a drug-resistant subpopulation At the transcript level, the cisplatin + DEAB-resistant cells showed upregulated mRNA expression levels for ALDH1A2, ALDH1A3 isozymes and CD44 indicating the involvement of these markers in conferring chemoresistance in both ALDHhigh and ALDHlow fractions of the three MPM cell lines Conclusions: Our study shows that ALDHhigh CD44+ cells are implicated in conveying tolerance to cisplatin in the three MPM cell lines The combined use of CD44 and ALDH widens the window for identification and targeting of a drug-resistant population which may improve the current treatment modalities in mesothelioma Keywords: Cancer stem cells, Malignant pleural mesothelioma, Chemoresistance, ALDH, CD44 Background Malignant pleural mesothelioma is an aggressive tumour with a very poor prognosis [1] The combination of pemetrexed and cisplatin is considered the front-line regimen for this disease, yielding a response rate of 41% and a median survival of 12.1 months [2] Despite continuous efforts to implement new therapeutic modalities, none of these, have prolonged patient survival primarily due to chemoresistance [1] It has been hypothesized that tumour relapse may be associated with the drug * Correspondence: ralph.schmid@insel.ch Department of Clinical Research, Division of General Thoracic Surgery, University Hospital Berne, Berne, Switzerland resistance of cancer stem cells; a rare cell population with the exclusive ability to self- renew and maintain a tumour [3] Hence, the identification and complete elimination of these cells presents an ultimate goal in MPM therapy Current studies have identified ALDH and CD44 as putative CSC markers which exhibit high chemoresistive properties in solid cancers; thus, rendering them as potential indicators of drug tolerance in MPM Aldehyde dehydrogenase enzymes are a family of intracellular enzymes that are involved in cellular proliferation, differentiation, detoxification, and drug resistance through the oxidation of cellular aldehydes [4,5] Certain ALDH isozymes are upregulated in tumour cells © 2014 Cortes-Dericks et al.; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Cortes-Dericks et al BMC Cancer 2014, 14:304 http://www.biomedcentral.com/1471-2407/14/304 resulting in decreased cellular sensitivity to cyclophosphamides and some oxazaphosphorines leading to decreased chemotherapeutic effect In leukemia and lung cancer cell lines overexpression of ALDH1A2a and ALDH2 increased cell proliferation and resistance to 4hydroperoxicyclophosphamide and doxorubicin indicating a role for the ALDH isozymes in drug resistance [6] The modulation of ALDH activity has been a central subject of research to improve the efficacy of conventional chemotherapeutic drugs Studies have shown that in small cell lung cancer, lung adenocarcinoma, leukemia and lung cancer cell lines the ALDH-dependent chemoresistance can be inhibited by DEAB or siRNA that conferred sensitivity to drug treatments [7-9] CD44, a transmembrane receptor for hyaluronan is associated with aggressive tumour growth, metastasis and resistance to therapy [10,11] In combination with other surface markers (e.g., ALDH and CD24), CD44 can discriminate between various cancer subsets In solid cancers the ALDHhigh/CD44+ subpopulation has been shown to possess stem cell-like properties that convey radio- and chemoresistance [12-14] ALDH+/CD44+ subpopulation can be sensitized by selective inhibition of ALDH activity using DEAB or all-trans retinoic acid, ATRA in breast cancer [14] As a single marker, CD44 is currently considered as a putative CSC indicator in human carcinomas including cancer of the lung In NSCLC cell lines, sorted CD44+ cells that bear stem cell-like properties conferred more resistance to cisplatin exhibiting lower apoptotic levels compared with CD44− cells [15] Despite the current evidence linking ALDH and CD44 to drug resistance in solid tumours, the variability in the different studies still warrants further investigation to delineate the present roles of these potential CSC markers Here, we sought to investigate whether ALDH can select for a drug-resistant subpopulation in three MPM cell lines We also assessed whether the ALDHhigh cells were associated with CD44, thus broadening the spectrum for identification of a drug-tolerant subpopulation in MPM The specific selection of a chemoresistant subpopulation using ALDH and CD44 may serve as a potential therapeutic target that may be employed as adjuvant therapy to the current standard treatment modalities in MPM Methods Cell culture The H28 and H2052 mesothelioma cell lines (LCD Promochem, France) were maintained in RPMI 1640 (PAA, Austria) containing 10% fetal bovine serum, FBS (PAA, Austria) and 1% penicillin/streptomycin solution (Invitrogen, Switzerland) ACC-Meso-4 cell line was purchased from Riken Cell Bank, Resource No: RBRC-RCB2293 (Ibaraki, Japan) and cultured using the above-mentioned culture Page of 13 medium Cells were cultured at 37°C, 95% humidity and 5% C02 The general information issued by the providers of the three MPM cell lines does not have data on drug resistance to cisplatin Sphere formation Single-cell preparations of parental and ALDH-sorted MPM cell lines were resuspended in an appropriate amount of sphere-forming medium (RPMI1640 supplemented with 20 ng/ml EGF and bFGF, [Invitrogen, Switzerland]; μg/ml insulin, [Sigma, Germany]; ml B27, [Invitrogen, Switzerland] and 1% penicillin/streptomycin solution) For all cell lines, x 103 cells/ml/well were seeded onto a 24-well ultra-low adherent plate (Costar, USA) Cells were incubated at 37°C, 95% humidity and 5% C02 for 7–14 days The documentation of images and evaluation of sphere-forming efficiency were performed on day Sphere-forming efficiency (%) was determined by dividing the number of spheres formed by the original number of seeded cells The quotient was then multiplied by 100 [16] Images were taken with Leica DMI 4000B at 5x magnification Drug treatment Drug resistance to cisplatin of mesothelioma cells were assessed by exposure to the IC50 values obtained for the non-sorted and ALDH-sorted cells for each of the three MPM cell lines For the determination of IC50, a dilution series of 2-fold increments of cisplatin (0–256 μM Cisplatin, CDDP, Bristol Myers Squibb, Switzerland) were prepared in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin Cells at a density of x 103cells/100 μl/well in 96-well plates were incubated in media with or without the addition of cisplatin Following a 48- and 72-hr incubation periods, culture media was aspirated, then replenished with XTT cell proliferation assay (Roche Chemicals, Switzerland) reagents After a 30-min incubation at 37°C, formazan production was measured spectrophotometrically at 450 nm Three independent experiments in triplicate were performed For cisplatin treatment, cells were cultured at x 104 cells/well in a 6-well plate (in three replicates) 48 hours prior to the addition of the previously determined IC50 of cisplatin for each cell line in RPMI 1640 containing 10% FBS and 1% penicillin/streptomycin solution Following the 48- and 72-h hour treatments at 37°C, cells were washed with PBS and harvested to perform the following: mRNA isolation, sphere formation assay and cell viability Pre-treatment of cells with 100 μM of ALDH inhibitor, DEAB (Sigma, Germany) was done for 48 h prior to cisplatin treatment [6,14] Cortes-Dericks et al BMC Cancer 2014, 14:304 http://www.biomedcentral.com/1471-2407/14/304 Aldefluor assay and flow cytometry The Aldefluor kit (Stem Cell Technologies, Canada) was used to identify the cells expressing ALDH activity Cells (0.5 – 1.0 x 106) were incubated in assay buffer containing ALDH substrate BODIPY-aminoacetaldehyde (BAAA) As negative control, an aliquot of the ALDH substratedtreated cells were immediately quenched with specific ALDH inhibitor, DEAB Both tubes were incubated for 45 at 37°C After incubation, cells were centrifuged and the pellets were resuspended with 500 μl of assay buffer prior to data acquisition using the green fluorescence channel of the LSR II flow cytometer (Becton Dickinson) This flow cytometry-based assay is termed as FACS analysis in this manuscript DEAB-treated cells served as control to set the ALDHhigh regions The same staining procedure was applied before sorting the cells with FACS Aria using FACS Diva software (Becton Dickinson) Cell purity was determined at each independent sorting After sorting of the ALDHhigh and ALDHlow fractions, these were immediately used in all experiments ALDHhigh CD44+ phenotype was assessed by immediately restaining the freshly-sorted ALDHhigh and ALDHlow cells with mouse anti-human CD44 APC-H7 (clone G44-26, BD Pharmingen, USA) and appropriate isotype control (mouse IgG2bK) for 30 on ice in the dark After washing with FACS buffer, data acquisition followed immediately Propidium iodide (PI) staining was used to exclude the non-viable cells FACS data were analysed with Flowjo software 7.2.5 (Treestar, Oregon, USA) RNA extraction and real-time quantitative PCR Cell cultures were collected in an appropriate amount of RNAprotect™ cell reagent (Qiagen, Germany) followed by total RNA extraction using RNeasy Kit ( Qiagen) according to manufacturer’s instructions Complementary DNA (cDNA) was synthesized by using the High capacity cDNA reverse transcription kit (Applied Biosystems, Rotkreuz, Switzerland) following the manufacturer’s instructions The mRNA levels of the housekeeping gene β2-microglobulin, β2M, and the target genes ALDH1A1, ALDH1A2, ALDH1A3, ALDH2 and CD44 were quantified with the commercially available TaqMan “Assay on Demand” primer/probes (β2M – Hs 99999903_m1; ALDH1A1 –Hs 00167445_m1; ALDH1A2- Hs 0018025_m1; ALDH1A3 –Hs 00167476_m1; ALDH2 – Hs 010007998_ml; CD44 –Hs01075861_m1) (Applied Biosystems; Rotkreuz, Switzerland) Twenty nanograms of resulting cDNAs were subjected to quantitative RT-PCR, in a 10 μl final reaction volume and analyzed in triplicates Gene expression was detected using ABI 7900 sequence detection system The gene expression level of each target gene was normalized by the endogeneous gene, β2M Page of 13 and compared among cells by the ΔΔCT method Baseline and threshold for Ct calculation were set automatically with ABI Prism SDS 2.1 software Statistical analysis Statistical analyses were performed using Graph Pad Prism 5.0 software© (San Diego, Ca) Two-tailed Student’s t-test was used to compare groups One- or two-way ANOVA with Bonferroni post hoc test as appropriate was performed to compare the values of >2 groups The statistical significance was set at p